Irradiation in vitro discriminates between different O-2A progenitor cell subpopulations in the perinatal central nervous system of rats

The effects of X irradiation on oligodendrocyte-type-2-astrocyte (O-2A) progenitor cells derived from different regions of the perinatal central nervous system (CNS) of rats were investigated in vitro. The O-2A progenitor cells can differentiate into either oligodendrocytes or type-2 astrocytes. The depletion of these cells could lead to demyelination, seen as a delayed reaction after irradiation of the CNS in vivo. To quantify cell survival, O-2A progenitor cells were grown on monolayers of type-1 astrocytes. Monolayers of type-1 astrocytes stimulate O-2A progenitor cells to divide. O-2A progenitor cells were irradiated in vitro and clonogenic cell survival was measured. The O-2A progenitor cells derived from perinatal optic nerve were quite radiosensitive in contrast to O-2A progenitor cells derived from perinatal spinal cord and perinatal corpus callosum. Furthermore, O-2A progenitor cells derived from the optic nerve formed smaller colonies, with most colonies showing early differentiation into oligodendrocytes. In contrast, more than half of the colonies derived from corpus callosum did not show any differentiation after 2 weeks in vitro and kept growing. These differences support the view that perinatal O-2A progenitor cells derived from the optic nerve are committed progenitor cells while the O-2A progenitor cells derived from the perinatal corpus callosum and the perinatal spinal cord have more stem cell properties.     

Cooperation between PDGF and FGF converts slowly dividing O-2Aadult progenitor cells to rapidly dividing cells with characteristics of O- 2Aperinatal progenitor cells

We have shown previously that oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells isolated from adult rat optic nerves can be distinguished in vitro from their perinatal counterparts on the basis of their much slower rates of division, differentiation, and migration when grown in the presence of cortical astrocytes or PDGF. This behavior is consistent with in vivo observations that there is only a modest production of oligodendrocytes in the adult CNS. As such a behavior is inconsistent with the likely need for a rapid generation of oligodendrocytes following demyelinating damage to the mature CNS, we have been concerned with identifying in vitro conditions that allow O-2Aadult progenitor cells to generate rapidly large numbers of progeny cells. We now provide evidence that many slowly dividing O-2Aadult progenitor cells can be converted to rapidly dividing cells by exposing adult optic nerve cultures to both PDGF and bFGF. In addition, these O-2Aadult progenitor cells appear to acquire other properties of O-2Aperinatal progenitor cells, such as bipolar morphology and high rate of migration. Although many O-2Aadult progenitor cells in cultures exposed to bFGF alone also divide rapidly, these cells are multipolar and migrate little in vitro. Oligodendrocytic differentiation of O-2Aadult progenitor cells, which express receptors for bFGF in vitro, is almost completely inhibited in cultures exposed to bFGF or bFGF plus PDGF. As bFGF and PDGF appear to be upregulated and/or released after injury to the adult brain, this particular in vitro response of O-2Aadult progenitor cells to PDGF and bFGF may be of importance in the generation of large numbers of new oligodendrocytes in vivo following demyelination.     

FGF modulates the PDGF-driven pathway of oligodendrocyte development

PDGF promotes the growth of oligodendrocyte type-2 astrocyte (O-2A) glial progenitor cells and allows their timely differentiation into oligodendrocytes, the CNS myelin-forming cells. We demonstrate that basic FGF is a potent mitogen for brain O-2A progenitor cells, but blocks their differentiation into oligodendrocytes. Treatment with basic FGF also influences the level of expression of PDGF receptors on O-2A progenitor cells. These cells express only the alpha chain PDGF receptor, and the levels of PDGF alpha receptors decrease as the cells differentiate. In contrast, basic FGF maintains a high level of functionally responsive PDGF alpha receptors in O-2A progenitors. Thus basic FGF activates a signaling pathway that can positively regulate PDGF receptors in O-2A progenitor cells. In this way basic FGF or an FGF-like factor may modulate the production of myelin-forming cells in the CNS.     

Coexistence of perinatal and adult forms of a glial progenitor cell during development of the rat optic nerve

We have studied the developmental appearance of the O-2A(adult) progenitor cell, a specific type of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell that we have identified previously in cultures prepared from the optic nerves of adult rats. O-2A(adult) progenitors differ from their counterparts in perinatal animals (O-2A perinatal progenitor cells) in antigenic phenotype, morphology, cell cycle time, rate of migration, time course of differentiation into oligodendrocytes or type-2 astrocytes and sensitivity to the lytic effects of complement in vitro. In the present study, we have found that O-2A(adult) progenitor-like cells first appear in the developing optic nerve approximately 7 days after birth and that by 1 month after birth these cells appear to be the dominant progenitor population in the nerve. However, the perinatal-to-adult transition in progenitor populations is a gradual one and O-2A(adult) and O-2A perinatal progenitors coexist in the optic nerve for 3 weeks or more. In addition, cells derived from optic nerves of P21 rats express characteristic features of O-2adult and O-2A perinatal progenitors for extended periods of growth in the same tissue culture dish. Our results thus indicate that the properties that distinguish these two types of O-2A progenitors from each other are expressed in apparently identical environments. Thus, these cells must either respond to different signals present in the environment, or must respond with markedly different behaviours to the binding of identical signalling molecules.     

Identification of an adult-specific glial progenitor cell

We have found that glial progenitor cells isolated from the optic nerves of adult rats are fundamentally different from their counterparts in perinatal animals. In our studies on bipotential oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, we have seen that O-2Aadult progenitor cells can be distinguished from O-2Aperinatal progenitors by their morphology and antigenic phenotype, their much longer cell cycle time (65 h versus 18 h), slower rate of migration rate (4 microns h-1 versus 21 microns h-1), and their time course of differentiation into oligodendrocytes or type-2 astrocytes in vitro (less than or equal to 3 days versus greater than 5 days). At least some of the differences between O-2Aadult and O-2Aperinatal progenitor cells appear to be clearly related to the differing cellular requirements of the adult and perinatal central nervous system (CNS). The properties of the O-2Aadult progenitor cells may make these cells ideally suited for the needs of the adult CNS, where rapid exponential increases in the number of oligodendrocytes and O-2A progenitor cells would be inappropriate. However, the properties of the O-2Aadult progenitor cells are such that they may not be able to replace oligodendrocytes in sufficient numbers to repair extensive or recurrent damage in the adult brain, such as in patients suffering from the human demyelinating disease multiple sclerosis. Moreover, available information about other tissues suggests that the transition from perinatal to adult progenitor cell types may represent a developmental mechanism of general importance.     

Clonal analysis of oligodendrocyte development in culture: evidence for a developmental clock that counts cell divisions

The clonal development of oligodendrocytes was studied by culturing individual oligodendrocyte--type-2 astrocyte (O-2A) progenitor cells on monolayers of type-1 astrocytes, which stimulate O-2A progenitor cells to divide. Oligodendrocytes developed by a proliferative lineage in which clonal progeny differentiated together after a number of cell divisions. Most O-2A progenitor cells had similar cell cycle times (1-2 days), but their proliferative capacity varied greatly: some divided only once while others divided up to eight times before differentiating. sister cells behaved similarly when recultured separately on astrocyte monolayers. These findings are consistent with the cell-division-counting hypothesis previously proposed to explain the timing of oligodendrocyte differentiation. They also unambiguously establish the phenotype of O-2A progenitor cells in vitro and demonstrate that these cells respond directly to growth factors produced by type-1 astrocyte monolayers.     

Ion channel expression by white matter glia: the O-2A glial progenitor cell.

We describe electrophysiological properties of the O-2A glial progenitor cell in a new serum-free culture system. O-2A progenitors have many properties characteristic of neurons: they have glutamate-activated ion channels, express the neuronal form of the sodium channel, fire single regenerative potentials, and synthesize the neurotransmitter GABA by an alternative synthetic pathway. Nearly identical properties were observed in acutely isolated O-2A progenitors, indicating that this phenotype is not an artifact of culture. The O-2A did not express a simple subset of channel types found in its descendant cells, the type-2 astrocyte and oligodendrocyte, studied in the same culture system. During development, these electrophysiological properties may contribute to O-2A function in vivo.     

Repopulation of O-2A progenitor cells after irradiation of the adult rat optic nerve analyzed by an in vitro clonogenic assay.

In the central nervous system (CNS) O-2A (Oligodendrocyte type 2 Astrocyte) progenitor cells have been proposed as potential target cells, and their depletion by irradiation will cause demyelination. The extent and time course of repopulation of these glial stem cells were studied in the adult rat optic nerve after irradiation in vivo. The number of O-2A progenitor cells was measured quantitatively by an in vitro clonogenic assay. Although the CNS is typically a late-responding tissue, repopulation was initiated almost immediately after irradiation and after several weeks a plateau was reached that lasted up to 6 months. Single doses of 4-12 Gy of X rays caused a permanent reduction in the number of O-2A progenitor cells. An analysis of the colony size of O-2A progenitor cells showed a sustained reduction in the number of offspring of cells surviving a dose of 12 Gy. In addition, the colony size of unirradiated progenitors diminished with increasing age of the animals.     

Proliferation and differentiation potential of rat forebrain oligodendroglial progenitors both in vitro and in vivo

We have followed the development of the O-2A progenitor cell from the neonatal rat forebrain, both in dissociated cell culture and in cryostat sections, using immunocytochemical techniques employing a panel of antibodies that recognise the cells at different stages of their development. This included the monoclonal antibody LB1, which binds to the surface ganglioside GD3 expressed on O-2A progenitor cells. In secondary cultures enriched for O-2A progenitors maintained in a serum-free chemically defined medium, a large proportion of the cells are primed to differentiate into oligodendroglia and go on to express the oligodendroglial specific surface glycolipid galactocerebroside (GC) and then the myelin proteins CNP and MBP. However, a significant proportion of immature bipolar GD3+ cells remained after 6 days in secondary culture. It appears that not all the O-2A progenitors in our cultures differentiate immediately and some cells remain in an undifferentiated state and divide to replenish progenitor numbers. We have also identified in our cultures a small apolar GD3- cell, which when isolated differentiated into a GD3+ bipolar O-2A progenitor cell. We have termed this cell type a preprogenitor. The differentiation of this cell type into O-2A progenitors may be the source of the immature GD3+ cells present at the later stages of our secondary cultures. The proliferative profile of the cultures was studied using 5'bromo-2-deoxyuridine (BrdU) incorporation as an index of mitosis. Only the immature, bipolar O-2A progenitors were seen to divide at any time in serum-free culture. Neither the more mature multipolar O-2A cells nor the oligodendroglia were seen to divide. The developmental profile of the O-2A cells in the rat forebrain in vivo showed a largely similar progression to that in culture, with a time lag of at least 6 days between GD3 expression and the onset of myelination. BrdU incorporation studies in vivo also showed that the GD3+ progenitor cell is mitotic whereas the GC(+)-expressing oligodendroglia is not. We have shown that there are several significant alterations in the timing of antigen expression in both O-2A progenitors and oligodendroglia in vitro compared to that seen in vivo.     

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

The O-2Aadult progenitor cell: a glial stem cell of the adult central nervous system

Systematic comparison of the properties of oligodendrocytetype-2 astrocyte (O-2A) progenitor cells derived from optic nerves of perinatal and adult rats has revealed that these two populations differ in many fundamental properties. In particular, O-2A^perinatal progenitor cells are rapidly dividing cells capable of generating large numbers of oligodendrocytes over a relatively short time span. Oligodendrocyte differentiation generally occurs synchronously in all members of a clone, thus leading to elimination of that clone from the pool of dividing cells. However, some O-2A^perinatal progenitors are also capable of giving rise to O-2A^adult progenitors. These latter cells express many of the characteristics of stem cells of adult animals, including the capacity to undergo asymmetric division and differentiation. We suggest that precursors which function during early development give rise to terminally differentiated end-stage cells and to a second generation of precursors with properties more appropriate for later developmental stages. It is this second generation of precursors which express the properties of stem cells in adult animals, and we therefore further suggest that our work offers novel insights into the possible developmental origin of stem cells.     

Analysis of the cell-cell interactions that control type-2 astrocyte development in vitro

Oligodendrocytes and type-2 astrocytes develop sequentially from O-2A progenitor cells in the rat CNS. We have reproduced this sequential development in a simplified, serum-free in vitro system: in cultures of newborn optic nerve cells treated with platelet-derived growth factor to maintain O-2A progenitor cell proliferation, progenitor cells differentiate into oligodendrocytes during the first week in vitro and into type-2 astrocytes during the second week. Thus all of the signals needed for type-2 astrocyte development are made by serum-free optic nerve cultures, indicating that neurons are not required. By manipulating the cellular composition of the cultures, we provide evidence that type-2 astrocyte development does not depend on oligodendrocytes, but instead requires non-O-2A lineage cells, which are also responsible for timing this development.     

A stochastic model to analyze clonal data on multi-type cell populations

This article presents a stochastic model designed to analyze experimental data on the development of cell clones composed of two (or more) distinct types of cells. The proposed model is an extension of the traditional multi-type Bellman-Harris branching stochastic process allowing for nonidentical time-to-transformation distributions defined for different cell types. A simulated pseudo likelihood method has been developed for the parametric statistical inference from experimental data on cell clones under the proposed model. The method uses simulation-based approximations of the means and the variance-covariance matrices of cell counts. The proposed estimator for the vector of unknown parameters is strongly consistent and asymptotically normal under mild regularity conditions, while its variance-covariance matrix is estimated by the parametric bootstrap. A Monte Carlo Wald test is proposed for the test of hypotheses. Finite sample properties of the estimator have been studied by computer simulations. The model and associated methods of parametric inference have been applied to the analysis of proliferation and differentiation of cultured O-2A progenitor cells that play a key role in the development of the central nervous system. It follows from this analysis that the time to division of the progenitor cell and the time to its differentiation (into an oligodendrocyte) are not identically distributed. This biological finding suggests that a molecular event determining the type of cell transformation is more likely to occur at the start rather than at the end of the mitotic cycle.     

Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell.

Optic nerves of neonatal rats contain a bipotential glial progenitor cell which can be induced by tissue culture conditions to differentiate into either an oligodendrocyte (the myelin-forming cell of the CNS) or a type 2 astrocyte (an astrocyte population found only in the myelinated tracts of the CNS). In our previous studies most oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells differentiated within 3 days in vitro with relatively little division of the progenitors or their differentiated progeny. We have now found that the O-2A progenitors are stimulated to divide in culture by purified populations of type 1 astrocytes, another glial cell-type found in the rat optic nerve. This cell-cell interaction appears to be mediated by a soluble factor(s) and results in the production of large numbers of both progenitor cells and oligodendrocytes. As type 1 astrocytes are the major glial cell-type in the optic nerve when oligodendrocytes first begin to be produced in large numbers in vivo, our results suggest that this astrocyte subpopulation may play an important role in expanding the oligodendrocyte population during normal development.     

IGF-I synergizes with FGF-2 to stimulate oligodendrocyte progenitor entry into the cell cycle

Secreted peptide growth factors are critical extracellular signals that interact to promote the proliferation, differentiation, and survival of progenitor cells in developing tissues. IGF-I signaling through the IGF type I receptor provides a mitogenic signal for numerous cell types, including stem and progenitor cells. We have utilized the O-2A oligodendrocyte progenitor to study the mechanism of IGF-I mitogenic actions since these progenitors respond to IGF-I in vitro, and gene targeting studies in mice have demonstrated that IGF-I is essential for normal oligodendrocyte development in vivo. The goal of this study was to elucidate the mechanism by which IGF-I promotes the proliferation of oligodendrocyte progenitors in the context of other mitogens critical for their proliferation. Results presented here show that IGF-I significantly amplified the actions of FGF-2 and PDGF to promote DNA synthesis in O-2A progenitors. Investigation of cell cycle kinetics revealed that IGF-I had no significant effect on the rate of cell cycle progression. Instead, IGF-I promoted increased recruitment of O-2A progenitors into the S phase of the cell cycle. These studies support a role for IGF-I as a cell cycle progression factor for progenitor cells.     

Development and regeneration in the central nervous system

As part of our attempts to understand principles that underly organism development, we have been studying the development of the rat optic nerve. This simple tissue is composed of three glial cell types derived from two distinct cellular lineages. Type-1 astrocytes appear to be derived from a monopotential neuroepithelial precursor, whereas type-2 astrocytes and oligodendrocytes are derived from a common oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell. Type-1 astrocytes modulate division and differentiation of O-2A progenitor cells through secretion of platelet-derived growth factor, and can themselves be stimulated to divide by peptide mitogens and through stimulation of neurotransmitter receptors. In vitro analysis indicates that many dividing O-2A progenitors derived from optic nerves of perinatal rats differentiate symmetrically and clonally to give rise to oligodendrocytes, or can be induced to differentiate into type-2 astrocytes. O-2Aperinatal progenitors can also differentiate to form a further O-2A lineage cell, the O-2Aadult progenitor, which has properties specialized for the physiological requirements of the adult nervous system. In particular, O-2Aadult progenitors have many of the features of stem cells, in that they divide slowly and asymmetrically and appear to have the capacity for extended self-renewal. The apparent derivation of a slowly and asymmetrically dividing cell, with properties appropriate for homeostatic maintenance of existing populations in the mature animal, from a rapidly dividing cell with properties suitable for the rapid population and myelination of central nervous system (CNS) axon tracts during early development, offers novel and unexpected insights into the possible origin of self-renewing stem cells and also into the role that generation of stem cells may play in helping to terminate the explosive growth of embryogenesis. Moreover, the properties of O-2Aadult progenitor cells are consistent with, and may explain, the failure of successful myelin repair in conditions such as multiple sclerosis, and thus seem to provide a cellular biological basis for understanding one of the key features of an important human disease.     

Oligodendrocyte precursor cells from different brain regions express divergent properties consistent with the differing time courses of myelination in these regions

Different CNS regions exhibit different temporal patterns of oligodendrocyte generation and myelinogenesis. Characterization of oligodendrocyte-type-2 astrocyte progenitor cells (here abbreviated as O-2A/OPCs) isolated from different regions indicates these developmental patterns are consistent with properties of the specific O-2A/OPCs resident in each region. Marked differences were seen in self-renewal and differentiation characteristics of O-2A/OPCs isolated from cortex, optic nerve and optic chiasm. In conditions where optic nerve-derived O-2A/OPCs generated oligodendrocytes within 2 days, oligodendrocytes arose from chiasm-derived cells after 5 days and from cortical O-2A/OPCs only after 7-10 days. These differences, which appear to be cell-intrinsic (and may be related to intracellular redox state), were manifested both in reduced percentages of clones producing oligodendrocytes and in a lesser representation of oligodendrocytes in individual clones. In addition, responsiveness of optic nerve-, chiasm- and cortex-derived O-2A/OPCs to thyroid hormone (TH) and ciliary neurotrophic factor (CNTF), well-characterized inducers of oligodendrocyte generation, was inversely related to the extent of self-renewal observed in basal division conditions. Our results demonstrate hitherto unrecognized complexities among the precursor cells thought to be the immediate ancestors of oligodendrocytes, and suggest that the properties of these different populations may contribute to the diverse time courses of myelination in different CNS regions.